Chronic itch is a presenting sign in numerous diseases associated with the skin, the immune, and the nervous systems. Chronic itch is largely resistant to conventional antihistamines, and few effective therapies are available. We have previously uncovered a small subset of itch- specific neurons which express gastrin-releasing peptide receptor (GRPR), an itch receptor, in the spinal cord. GRPR+ neurons are required for pruritoceptive but not for nociceptive transmission. We have also begun to elucidate the function of neuromedin B receptor (NMBR), another itch receptor in the spinal cord. Our goal is to investigate fundamental mechanisms of itch transmission. We will take advantage of several unique mouse lines to examine the dorsal horn itch circuitry at the single cell level. Aim 1 will determine neurochemical, anatomic and electrophysiological properties of GRPR neurons. We will examine the neurotransmitter phenotype of GRPR neurons and whether GRPR neurons are projection or interneurons by retrograde labeling. We will also examine electrophysiological properties of GRPR neurons using whole cell patch-clamp recording. Aim 2 will determine the synaptic mechanisms underlying GRP-mediated itch transmission. We will test the hypothesis that GRP-evoked excitation of GRPR neurons is mediated by modulation of ionotropic glutamate receptors (iGluRs) and GRP-dependent mechanism is differentially involved in pruritogens-induced pruriceptive transmission. We will also determine the role of glutamatergic input in this process and examine whether nonhistaminergic and histaminergic pruritogen act through GRP and iGluRs. Aim 3 will determine molecular properties and function of itch-sensing neurons in chronic itch condition. We will determine whether ectopic GRPR expression induced in chronic itch condition is dependent on primary GRPR neurons by cell ablation approach. Moreover, we will characterize the electric properties of GRPR neurons in chronic itch condition. Finally, we will examine the role of NMBR neurons in itch sensation using NMB-saporin approach.